Isolated transcription factor for an alpha-amylase promoter in filamentous fungi

ABSTRACT

The present invention relates to a transcription factor found in filamentous fungi, especially in Aspergillii, DNA sequences coding for said factor, its transformation into and expression in fungal host organisms, and the use of said factor in such hosts for increasing the expression of a polypeptide of interest being produced by said host.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 09/197,814filed Nov. 23, 1998, now U.S. Pat. No. 6,316,220, which is acontinuation of PCT/DK97/00305 filed Jul. 7, 1997 (the internationalapplication was published under PCT Article 21(2) in English) andclaims, under 35 U.S.C. 119, priority of Danish application no. 0740/96filed Jul. 5, 1996, the contents of which are fully incorporated hereinby reference.

FIELD OF THE INVENTION

The present invention relates to a transcription factor found infilamentous fungi, especially in Aspergillii, DNA sequences coding forsaid factor, its transformation into and expression in fungal hostorganisms, and the use of said factor in such hosts for increasing theexpression of a polypeptide of interest being produced by said host.

BACKGROUND OF THE INVENTION

Transcription factors are well known proteins involved in the initiationof transcription. They have been studied intensively in many differentorganisms and have also been described in fungi. Dhawale and Lane (NAR(1993) 21 5537-5546) have recently compiled the transcription factorsfrom fungi, including the filamentous fungi.

Many of the transcription factors are regulatory proteins; they bind tothe promoter DNA and either activate or repress transcription as aresponse to stimuli to the cell.

The expression of the α-amylase gene in A. oryzae is regulated inresponse to the available carbon source. The gene is expressed at itsmaximum when the organism is grown on starch or maltose (Lachmund et al.(1993) Current Microbiology 26 47-51; Tada et al. (1991) Mol. Gen. Genet229 301-306). The expression of α-amylase is regulated at thetranscriptional level as shown by Lachmund et aL (supra), which stronglysuggests that transcription factors are involved in the regulation, butso far no gene for such a factor has been identified.

The promoter of the α-amylase gene has been studied by deletion analysis(Tada et al. (1991) Agric. Biol. Chem. 55 1939-1941; Tsuchiya et al.(1992) Biosci. Biotech. Biochem. 56 1849-1853; Nagata et al. (1993) Mol.Gen. Genet 237 251-260). The authors of these papers propose that aspecific sequence of the promoter is responsible for the maltoseinduction. Nagata et al. (supra) used this sequence as a probe in a gelshift experiment to see whether any proteins from A. nidulans nuclearextracts were able to bind to the promoter sequence. Three such proteinswere found, but no involvement of these proteins in expression wasshown. None of the proteins have been purified or identified by othermeans. Their genes likewise remain unknown.

SUMMARY OF THE INVENTION

The present invention relates to a transcription factor regulating theexpression of the α-amylase promoter in filamentous fungi.

Accordingly, in a first aspect the invention relates to a DNA constructcomprising a DNA sequence encoding a transcription factor of theinvention, which DNA sequence comprises:

a) the transcription factor encoding part of the DNA sequence clonedinto plasmid pToC320 present in E. coli ToC1058, DSM 10666, or

b) an analogue of the DNA sequence defined in a), which

i) is at least 60% homologous with the DNA sequence defined in a), or

ii) hybridizes with the same nucleotide probe as the DNA sequencedefined in a), or

iii) encodes a transcription factor which is at least 50% homologouswith the transcription factor encoded by a DNA sequence comprising theDNA sequence defined in a), or

iv) encodes a transcription factor which is immunologically reactivewith an antibody raised against the purified transcription factorencoded by the DNA sequence defined in a), or

v) complements the mutation in ToC879, i.e. enables ToC879 to grow oncyclodextrin and produce lipase when transformed with said DNA sequence.

The full length genomic DNA sequence encoding a transcription factor hasbeen derived from a strain of the filamentous fungus Aspergillus oryzaeand has been cloned into plasmid pToC320 present in E. coli ToC1058, DSM10666.

Said transcription factor encoding DNA sequence harboured in pToC320,DSM 10666, is believed to have the same sequence as that presented inSEQ ID NO: 1 and SEQ ID NO: 2. Accordingly, whenever reference is madeto the transcription factor encoding part of the DNA sequence clonedinto plasmid pToC320 present in DSM 10666 such reference is alsointended to include the transcription factor encoding part of the DNAsequence presented in SEQ ID NO: 1 and SEQ ID NO: 2.

Accordingly, the terms “the transcription factor encoding part of theDNA sequence cloned into plasmid pToC320 present in DSM 10666” and “thetranscription factor encoding part of the DNA sequence presented in SEQID NO: 1 and SEQ ID NO: 2” may be used interchangeably.

In further aspects the invention provides an expression vectorharbouring the DNA construct of the invention, a cell comprising saidDNA construct or said expression vector and a method of producing apeptide exhibiting transcription factor activity, which method comprisesculturing said cell under conditions permitting the production of thetranscription factor.

Such a transcription factor of the invention will typically originatefrom a filamentous fungus.

The term “filamentous fungus” is intended to include the groupsPhycomycetes, Zygomycetes, Ascomycetes, Basidiomycetes and fungiimperfecti, including Hyphomycetes such as the genera Aspergillus,Penicillium, Trichoderma, Fusarium and Humicola.

The invention also relates to a method of producing a filamentous fungalhost cell comprising the introduction of a DNA fragment coding for anysuch factor into a filamentous fungus wherein an α-amylase promoter or aco-regulated promoter regulates the expression of a polypeptide ofinterest in a manner whereby said factor will be expressed in saidfungus.

In a further aspect the invention relates to a method of producing apolypeptide of interest, the expression of which is regulated by anα-amylase promoter or a co-regulated promoter, comprising growing afilamentous fungal host cell as described above under conditionsconducive to the production of said factor and said polypeptide ofinterest, and recovering said polypeptide of interest.

Finally the invention relates to the use of said factor for regulatingthe expression of a polypeptide of interest in a filamentous fungus.

In this context, regulation means to change the conditions under whichthe factor of the invention is active. This could mean different pH,substrate, etc. regimes, whereby the resulting effect is an improvedregulation of the expression of the protein of interest.

Furthermore, regulation also comprises events occurring in the growthphase of the fungus during which the transcription factor is active.Depending on the circumstances, both advancing and/or postponing thephase wherein the factor is active may enhance the expression and thusthe yield.

In addition, using standard procedures known in the art, the specificDNA sequences involved in the binding of a transcription factor may beidentified, thereby making it possible to insert such sequences intoother promoters not normally regulated by the factor and enabling thosepromoters to be under the regulation of said factor.

BRIEF DESCRIPTION OF THE TABLES AND DRAWING

FIG. 1 shows the structure of the plasmid pMT1657, the construction ofwhich is described in Example 1;

FIG. 2 shows the structure of the plasmid pToC316, the construction ofwhich is described in Example 1;

FIG. 3 shows the structure of the plasmid pToC320, the construction ofwhich is described in Example 1;

FIG. 4 shows the structure of the plasmids pToC342 and pToC359, theconstruction of which are described in Example 3;

FIG. 5 shows the structure of the plasmid pToC298, the construction ofwhich is described in Example 4;

FIG. 6 shows the results of lipase production by a p960 transformant ofA. oryzae IFO4177 cultured in YP media containing 2% glucose (-¦-) or10% glucose (-?-), in comparison to ToC1075 cultured in YP mediacontaining 2% glucose (--) or 10% glucose (-⋄-) and described in Example4;

FIG. 7 shows the results of lipase production by ToC1139 cultured in YPmedia containing 2% glucose (-¦-) or 10% glucose (-?-), in comparison toToC1075 cultured in YP media containing 2% glucose (--) or 10% glucose(-⋄-) and described in Example 4; and

FIG. 8 shows the autoradiograph results of A. niger DNA digested withthe following restriction enzymes: lane 2, XbaI; lane 3, XmaI; lane 4,Sall; lane 5, HindIII; lane 6, EcoRI; lane 7, BgIII; lane 8, BamHI;lanes 1 and 9 contain ³²P-labelled I DNA digested with BstEII. Theexperiment is described in Example 5.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect the invention relates to a DNA construct comprising aDNA sequence encoding a transcription factor regulating an α-amylasepromoter, which DNA sequence comprises

a) the transcription factor encoding part of the DNA sequence clonedinto plasmid pToC320 present in E. coli ToC1058, DSM 10666 (SEQ ID NOS:1 and 2), or

b) an analogue of the DNA sequence defined in a), which

i) is at least 60% homologous with the DNA sequence defined in a), or

ii) hybridizes with the same nucleotide probe as the DNA sequencedefined in a), or

iii) encodes a transcription factor which is at least 50% homologouswith the transcription factor encoded by a DNA sequence comprising theDNA sequence defined in a), or

iv) encodes a transcription factor which is immunologically reactivewith an antibody raised against the purified transcription factorencoded by the DNA sequence defined in a), or

v) complements the mutation in ToC879, i.e. enables ToC879 to grow oncyclodextrin and produce lipase when transformed with said DNA sequence.

As defined herein, a DNA sequence analogous to the transcription factorencoding part of the DNA sequence cloned into plasmid pToC320 present inE. coli ToC1058, DSM 10666, is intended to indicate any DNA sequenceencoding a transcription factor regulating an α-amylase promoter, whichtranscription factor has one or more of the properties cited under(i)-(v) above.

The analogous DNA sequence may be isolated from a strain of thefilamentous fungus A. oryzae producing the transcription factor, oranother or related organism and thus, e.g. be an allelic or speciesvariant of the transcription factor encoding part of the DNA sequencecloned into plasmid pToC320 present in DSM 10666.

Alternatively, the analogous sequence may be constructed on the basis ofthe DNA sequence presented as the transcription factor encoding part ofSEQ ID NO: 1 and SEQ ID NO: 2, e.g. be a sub-sequence thereof, and/or byintroduction of nucleotide substitutions which do not give rise toanother amino acid sequence of the transcription factor encoded by theDNA sequence, but which corresponds to the codon usage of the hostorganism intended for production of the transcription factor, or byintroduction of nucleotide substitutions which may give rise to adifferent amino acid sequence.

When carrying out nucleotide substitutions, amino acid residue changesare preferably of a minor nature, that is conservative amino acidresidue substitutions that do not significantly affect the folding oractivity of the protein, small deletions, typically of one to about 30amino acid residues; small amino- or carboxyl-terminal extensions.

Examples of conservative substitutions are within the group of basicamino acids (such as arginine, lysine, histidine), acidic amino acids(such as glutamic acid and aspartic acid), polar amino acids (such asglutamine and asparagine), hydrophobic amino acids (such as leucine,isoleucine, valine), aromatic amino acids (such as phenylalanine,tryptophan, tyrosine) and small amino acids (such as glycine, alanine,serine, threonine, methionine). For a general description of nucleotidesubstitution, see e.g. Ford, et aL., (1991), Protein Expression andPurification 2, 95-107.

It will be apparent to persons skilled in the art that suchsubstitutions can be made outside the regions critical to the functionof the molecule and still result in an active transcription factor.Amino acid residues essential to the activity of the transcriptionfactor encoded by a DNA construct of the invention, and thereforepreferably not subject to substitution, may be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (cf. e.g. Cunningham and Wells, (1989),Science 244 1081-1085). In the latter technique mutations are introducedat every residue in the molecule, and the resultant mutant molecules aretested for biological activity (i.e. transcription factor regulating anα-amylase promoter) to identify amino acid residues that are critical tothe activity of the molecule.

The homology referred to in (i) above is determined as the degree ofidentity between the two sequences indicating a derivation of the onesequence from the other. The homology may suitably be determined bymeans of computer programs known in the art such as GAP provided in theGCG program package (Needleman, S. B. and Wunsch, C. D., (1970), Journalof Molecular Biology 48 443-453). Using GAP with the following settingsfor DNA sequence comparison: GAP creation penalty of 5.0 and GAPextension penalty of 0.3, the coding region of the DNA sequence exhibitsa degree of identity preferably of at least 60%, more preferably atleast 70%, more preferably at least 80%, more preferably at least 90%,more preferably at least 95% with the transcription factor encoding partof the DNA sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2.

The hybridization referred to in (ii) above is intended to indicate thatthe analogous DNA sequence hybridizes to the same probe as the DNAsequence encoding the transcription factor under certain specifiedconditions, which are described in detail in the Materials and Methodssection hereinafter. The oligonucleotide probe to be used is the DNAsequence corresponding to the transcription factor encoding part of theDNA sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a fragmentthereof.

The homology referred to in (iii) above is determined as the degree ofidentity between the two sequences indicating a derivation of the firstsequence from the second. The homology may suitably be determined bymeans of computer programs known in the art such as GAP provided in theGCG program package (Needleman, S. B. and Wunsch, C. D., supra). UsingGAP with the following settings for transcription factor sequencecomparison: GAP creation penalty of 3.0 and GAP extension penalty of0.1, the transcription factor encoded by an analogous DNA sequenceexhibits a degree of identity preferably of at least 50%, morepreferably at least 60%, more preferably at least 70%, even morepreferably at least 80%, especially at least 90% with the transcriptionfactor encoded by a DNA construct comprising the transcription factorencoding part of the DNA sequence shown in SEQ ID NO: 2, e.g. with theamino acid sequence SEQ ID NO: 3.

In connection with property (iv) the immunological reactivity may bedetermined by the method described in the Materials and Methods sectionhereinafter.

In relation to the property (v) the complementation method is describedin Example 1 herein.

The DNA sequence encoding a transcription factor of the invention can beisolated from the strain Aspergillus oryzae IFO 4177 using standardmethods e.g. as described by Sambrook, et al., (1989) Molecular Cloning:A Laboratory Manual. Cold Spring Harbor Lab.; Cold Spring Harbor, N.Y.

General RNA and DNA isolation methods are also disclosed in WO 93/11249and WO 94/14953, the contents of which are hereby incorporated byreference. A more detailed description of the complementation method isgiven in Example 1 herein.

Alternatively, the DNA encoding a transcription factor of the inventionmay, in accordance with well-known procedures, be conveniently isolatedfrom a suitable source, such as any of the below mentioned organisms, byuse of synthetic oligonucleotide probes prepared on the basis of a DNAsequence disclosed herein. For instance, a suitable oligonucleotideprobe may be prepared on the basis of the transcription factor encodingpart of the nucleotide sequences presented as SEQ ID NO: 1 or anysuitable subsequence thereof, or on the basis of the amino acid sequenceSEQ ID NO: 3.

The invention relates specifically to a transcription factor regulatingthe expression of the α-amylase promoter in a filamentous fungus, whichfactor as indicated in Example 2 may even regulate the expression ofother genes.

In this context the expression “filamentous fungus” is intended toinclude the groups Phycomycetes, Zygomycetes, Ascomycetes,Basidiomycetes and fungi imperfecti, including Hyphomycetes such as thegenera Aspergillus, Penicillium, Trichoderma, Fusarium and Humicola.

In this context the expression “α-amylase promoter” means a sequence ofbases immediately upstream from an α-amylase gene which RNA polymeraserecognises and binds to promoting transcription of the gene coding forthe α-amylase.

As indicated, transcription factors are known from many organisms and itis therefore expected that similar or corresponding factors may be foundoriginating from other fungi of the genera Aspergillus, Trichoderma,Penicillium, Fusarium, Humicola, etc., having an enhancing effect on theexpression of a polypeptide being under the regulation of amylasepromoters in any fungus belonging to any of these genera.

A comparison of the DNA sequence coding for the transcription factorregulating the α-amylase promoter has shown some degree of homology to atranscription factor (CASUCI) regulating the expression of glucosidasein Candida and to MAL63 of Saccharomyces cerevisiae as disclosed inKelly and Kwon-Chung, (1992) J. Bacteriol. 174 222-232.

It is at present contemplated that a DNA sequence encoding atranscription factor homologous to the transcription factor of theinvention, i.e. an analogous DNA sequence, may be obtained from othermicroorganisms. For instance, the DNA sequence may be derived by asimilar screening of a cDNA library of another microorganism, such as astrain of Aspergillus, Saccharomyces, Erwinia, Fusarium or Trichoderma.

An isolate of a strain of A. oryzae from which the gene coding for atranscription factor of the invention has been inactivated has beendeposited by the inventors according to the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure at the DSM, Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124Braunschweig, DEUTSCHLAND.

Deposit date: MAY 6, 1996 (May 6, 1996)

Depositor's ref.: ToC879=NN049238

DSM designation: Aspergillus oryzae DSM No.10671

The deposited strain Aspergillus oryzae DSM No.10671 can be used toisolate a transcription factor according to the invention from anystrain of Aspergillus oryzae and any other fungal strain having such agene by complementation as described hereinafter.

The expression plasmid pToC320 comprising the full length genomic DNAsequence encoding the transcription factor of the invention has beentransformed into a strain of E. coli resulting in the strain ToC1058,which has been deposited by the inventors according to the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purposes of Patent Procedure at the DSMZ, Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH., Mascheroder Weg 1b, D-38124Braunschweig, DEUTSCHLAND.

Deposit date: MAY 6, 1996 (May 6, 1996)

Depositor's ref.: ToC1058=NN049237

DSM designation: E. coli DSM No.10666

According to the invention, factors of this type originating from thespecies A. oryzae, A. niger, A. awamori, etc., especially A. oryzaeIFO4177 are preferred.

The transcription factor of the invention has been found not only to beinvolved in the regulation of the α-amylase promoter, but also in theregulation of the glucoamylase promoter from A. oryzae.

Especially, the invention comprises any factor having an amino acidsequence comprising one or more fragments or combinations of fragmentsof the amino acid sequence depicted as SEQ ID NO: 3.

Truncated forms of the transcription factor may also be active. Bytruncated forms are meant modifications of the transcription factorwherein N-terminal, C-terminal or one or more internal fragments havebeen deleted.

A further aspect of the invention relates to a DNA sequence coding forany of these factors.

In this aspect the invention especially comprises any DNA sequencecoding for one or more fragments of the amino acid sequence depicted asSEQ ID NO: 3.

More specifically the invention relates to a DNA sequence comprising oneor more fragments or a combination of fragments of the DNA sequencedepicted as SEQ ID NO: 1 and SEQ ID NO: 2.

According to a further aspect the invention relates to a method ofproducing a filamentous fungal host cell comprising the introduction ofany of the above mentioned DNA fragments into a filamentous funguswherein the α-amylase promoter or another co-regulated promoterregulates the expression of a polypeptide of interest in a mannerwhereby said factor will be expressed in said fungus.

The introduction of said DNA fragment may be performed by means of anywell known standard method for the introduction of DNA into afilamentous fungus, such as by use of an expression vector and hostcells as described below.

Therefore, the invention also provides a recombinant expression vectorcomprising the DNA construct of the invention.

The expression vector of the invention may be any expression vector thatis conveniently subjected to recombinant DNA procedures, and the choiceof vector will often depend on the host cell into which it is to beintroduced.

Thus, the vector may be an autonomously replicating vector, i.e. avector which exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g. a plasmid.Alternatively, the vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

In the expression vector, the DNA sequence encoding the transcriptionfactor should either also contain the expression signal normallyassociated with the transcription factor or should be operably connectedto a suitable promoter and terminator sequence. The promoter may be anyDNA sequence which shows transcriptional activity in the host cell ofchoice and may be derived from genes that are either homologous orheterologous to the host cell. The procedures used to ligate the DNAsequences coding for the transcription factor, the promoter and theterminator, respectively, and to insert them into suitable vectors arewell known to persons skilled in the art (cf., Sambrook, et al., supra).

Examples of suitable promoters for use in filamentous fungal host cellsare, for instance, the A. nidulans ADH3 promoter (McKnight, et al.(1985) The EMBO J. 4 2093-2099) or the tpiA promoter. Examples of otheruseful promoters are those derived from the gene encoding Aspergillusoryzae α-amylase, Aspergillus niger neutral alpha-amylase, Aspergillusniger acid stable alpha-amylase, Aspergillus niger, Aspergillus awamori,or Aspergillus. oryzae glucoamylase (gluA), A. oryzae alkaline protease(alp), A. oryzae nitrate reductase (niaD), Aspergillus oryzae triosephosphate isomerase (tpi), Aspergillus nidulans acetamidase, or anAspergillus promoter coding for an amino acid biosynthetic gene such asargB.

In yet another aspect the invention provides a host cell comprising theDNA construct of the invention and/or the recombinant expression vectorof the invention.

Preferably, the host cell of the invention is a eukaryotic cell, inparticular a fungal cell such as a yeast or filamentous fungal cell. Inparticular, the cell may belong to a species of Trichoderma, preferablyTrichoderma harzianum or Trichoderma reesei, or a species ofAspergillus, most preferably Aspergillus oryzae or Aspergillus niger.Fungal cells may be transformed by a process involving protoplastformation and transformation of the protoplasts followed by regenerationof the cell wall in a manner known per se. The use of Aspergillus as ahost microorganism is described in EP 238 023 (Novo Nordisk A/S), thecontents of which are hereby incorporated by reference. The host cellmay also be a yeast cell, e.g. a strain of Saccharomyces, in particularSaccharomyces cerevisiae, Saccharomyces kluyveri or Saccharomycesuvarum, a strain of Schizosaccharomyces sp., such as Schizosaccharomycespombe, a strain of Hansenula sp., Pichia sp., Yarrowia sp., such asYarrowia lipolytica, or Kluyveromyces sp., such as Kluyveromyces lactis.

The endogenous amyR gene of the host cell may be deleted or inactivatedby other means. The introduction of amyR control by a heterologouspromoter will then lead to a completely new scheme of regulation of thealpha-amylase promoter. If, for example, amyR is fused to the A. oryzaeniaD promoter, the alpha-amylase promoter will become inducible bynitrate. If, instead of the niaD promoter, a palC-regulated promoter isused, the activity of the alpha-amylase promoter will be regulated bypH.

The invention also comprises a method of producing a polypeptide ofinterest, whereby a host cell as described above is grown underconditions conducive to the production of said factor and saidpolypeptide of interest, and said polypeptide of interest is recovered.

The medium used to culture the transformed host cells may be anyconventional medium suitable for growing the host cells in question. Theexpressed polypeptide of interest may conveniently be secreted into theculture medium and may be recovered therefrom by well-known proceduresincluding separating the cells from the medium by centrifugation orfiltration, precipitating proteinaceous components of the medium bymeans of a salt such as ammonium sulphate, followed by chromatographicprocedures such as ion exchange chromatography, affinity chromatography,or the like.

According to the invention the method may be used to produce apolypeptide of interest that is a medicinal polypeptide, especially suchmedicinal polypeptides as growth hormone, insulin, blood clottingfactor, and the like.

The method of the invention may also be used for the production ofindustrial enzymes, such as proteases, lipases, amylases, glucoamylases,oxido reductases, carbohydrases, carbonyl hydrolases, cellulases,esterases, etc.

According to a further aspect of the invention said transcription factormay be used for enhancing the expression of a polypeptide of interest ina filamentous fungus, such as a fungus of the genus Aspergillus,Trichoderma, Penicillium, Fusarium, Humicola, etc., especially of thespecies A. oryzae, A. niger, A. awamori, etc., and specifically A.oryzae.

The transcription factor of the invention may thus be used to enhancethe expression of a medicinal polypeptide, such as growth hormone,insulin, blood clotting factor, etc.

Also, the expression of industrial enzymes, such as proteases, lipases,amylases, glucoamylases, oxido reductases, carbohydrases, carbonylhydrolases, cellulases, esterases, etc., may be enhanced by the use ofthe transcription factor of the invention.

The transcription factor may also be used to identify the sequences inthe alpha-amylase promoter to which it binds. For example, this could bedone by making a GST-fusion protein with the DNA binding domain of AmyR,such as the zinc finger, for production in E. coli. Such fusion proteinsmay be conveniently made using commercially available kits, for example,“The GST Gene Fusion Kit” from Pharmacia. The purified GST-fusionprotein can then be used in conventional in vitro techniques such as gelshift assays or DNA footprint analyses (Kulmburg, P., et al. (1992)Molecular and Cellular Biology 12 1932-1939; Lutfiyya, L. L., andJohnston, M. (1996) Molecular and Cellular Biology 16 4790-4797). Theidentification of the AmyR binding site will make it possible to insertthese sequences in other promoters not normally regulated by AmyR.

Materials and Methods

Hybridization

Suitable hybridization conditions for determining hybridization betweena nucleotide probe and an “analogous” DNA sequence of the invention maybe defined as described below. The oligonucleotide probe to be used isthe DNA sequence corresponding to the transcription factor encoding partof the DNA sequence shown in SEQ ID NO: 1, i.e. nucleotides1691..2676+2743..3193+3278..3653 in SEQ ID NO: 1, or a fragment thereof,e.g. nucleotides 1770-1800 in SEQ ID NO: 1.

Hybridization Conditions

Suitable conditions for determining hybridization between a nucleotideprobe and a homologous DNA or RNA sequence involves pre-soaking of thefilter containing the DNA fragments or RNA to hybridize in 5×SSC(standard saline citrate buffer) for 10 min, and prehybridization of thefilter in a solution of 5×SSC (Sambrook, et al., supra), 5×Denhardt'ssolution (Sambrook, et al., supra), 0.5% SDS and 100 μg/ml of denaturedsonicated salmon sperm DNA (Sambrook, et al., supra), followed byhybridization in the same solution containing a random-primed (Feinberg,A. P. and Vogelstein, B. (1983) Anal. Biochem. 132 6-13),³²P-dATP-labeled (specific activity>1×10⁹ cpm/μg) probe for 12 hours atca. 65° C. The filter is then washed two times for 30 minutes in 2×SSC,0.5% SDS at preferably not higher than 50° C., more preferably nothigher than 55° C., more preferably not higher than 60° C., morepreferably not higher than 65° C., even more preferably not higher than70° C., especially not higher than 75° C.

Molecules to which the nucleotide probe hybridizes under theseconditions are detected using a Phospho Image detector.

Immunological Cross-reactivity

Antibodies to be used in determining immunological cross-reactivity maybe prepared by use of a purified transcription factor. Morespecifically, antiserum against the transcription factor of theinvention may be raised by immunizing rabbits (or rodents) according tothe procedure described by N. Axelsen et al. in: A Manual ofQuantitative Immunoelectrophoresis, Blackwell Scientific Publications,1973, Chapter 23, or A. Johnstone and R. Thorpe, Immunochemistry inPractice, Blackwell Scientific Publications, 1982 (more specifically pp.27-31). Purified immunoglobulins may be obtained from the antisera, forexample by salt precipitation ((NH₄)₂ SO₄), followed by dialysis and ionexchange chromatography, e.g. on DEAE-Sephadex. Immunochemicalcharacterization of proteins may be done either by Outcherlonydouble-diffusion analysis (O. Ouchterlony in: Handbook of ExperimentalImmunology (D. M. Weir, ed.), Blackwell Scientific Publications, 1967,pp.655-706), by crossed immunoelectrophoresis (N. Axelsen et al., supra,Chapters 3 and 4), or by rocket immunoelectrophoresis (N. Axelsen etal., op cit., Chapter 2).

EXAMPLES Example 1

Cloning of the amyR Transcription Factor from A. oryzae

amyR was cloned by complementation of an A. oryzae mutant strain unableto express two different proteins both under control of the TAKA-amylasepromoter. The mutant A. oryzae strain ToC879 was made by mutagenesis ofa strain, SRe440, containing a lipase (HLL) encoding cDNA under controlof the TAKA promoter and one copy of the TAKA-amylase gene transcribedfrom its own promoter.

The mutant was identified and isolated by its amylase negative(amylase⁻) phenotype and subsequently shown to be lipase negative(lipase⁻) as well.

The strain ToC879 contains intact copies of both expression cassettes.The amylase⁻ phenotype makes ToC879 unable to grow on plates containing1% cyclodextrin as the sole carbon source, while the parent strainSRe440 will grow on such plates.

ToC879 has been deposited at DSM under the name DSM No.10671.

amyR was isolated by co-transforming ToC879 with an A. oryzae cosmidlibrary and an autonomously replicating pHelp1 based plasmid (D. Gems,I. L. Johnstone, and A. J. Clutterbuck (1991) Gene 98 61-67) carryingthe bar gene from Streptomyces hygroscopicus which confers resistance toglufosinate. The transformants were subjected to selection on platescontaining cyclodextrin as the sole carbon source and screened for aconcurrent reversion to the lipase⁺ phenotype.

The transforming DNA was rescued from colonies able to grow oncyclodextrin. Subcloning resulted in the isolation of a 4.3 kb DNAfragment able to complement both phenotypes of ToC879. The geneharboured on this fragment was named amyR.

Construction of the pHelp1 Derivative pMT1657Construction of the pHelp1Derivative pMT1657.Construction of the pHelp1 Derivative pMT1657.

A plasmid, pMT1612, was made by ligation (and subsequent transformationinto E. coli DH5a) of the following four fragments:

i) the E. coli vector pToC65 (described in EP 531 372) cut withSphI/XbaI,

ii) a PCR fragment (containing the A. nidulans amdS promoter) cut withSphI/BamHI,

iii) a 0.5 kb BamHI/XhoI fragment from pBP1T (B. Staubinger et al.,(1992) Fungal Genetics Newsletter 39 82-83) containing the bar gene, and

iv) a 0.7 kb XhoI/XbaI fragment from pIC AMG/Term (EP Application No.87103806.3) containing the A. niger glucoamylase transcriptionterminator.

The PCR fragment containing the amdS promoter was made using the plasmidpMSX-6B1 (M. E. Katz et al., (1990) Mol. Gen. Genet. 220 373-376) assubstrate DNA and the two oligonucleotides 4650 (SEQ ID NO: 4) and 4561(SEQ ID NO: 5) as primers.

4650: CTTGCATGCCGCCAGGACCGAGCAAG, SEQ ID NO: 4 4651:CTTGGATCCTCTGTGTTAGCTTATAG. SEQ ID NO: 5

pMSX-6B1 contains an amdS promoter up mutation called 1666. pMT1612 wascut with HindIII, dephosphorylated and ligated to a 5.5 kb HindIIIfragment from pHelp1 containing the AMA1 sequence. The resultingplasmid, pMT1657 is self-replicating in Aspergilli and can be selectedfor by increased resistance to glufosinate. pMT1657 is depicted in FIG.1, wherein PamdS represents the amdS promoter of fragment ii) above, barrepresents fragment iii) above, and Tamg represents fragment iv) above.

Construction of the Cosmid LibraryConstruction of the Cosmid Library

A cosmid library of Aspergillus oryzae was constructed essentiallyaccording to the instructions from the supplier of the “SuperCos1 cosmidvector kit” (Stratagene Cloning Systems, La Jolla Calif., USA).

Genomic DNA of A. oryzae IFO4177 was prepared from protoplasts made bystandard procedures (Christensen, T., et al. (1988) Biotechnology 61419-1422).

After isolation the protoplasts were pelleted by centrifugation at 2500rpm for 5 minutes in a Labofuge T (Heto); the pellet was then suspendedin 10 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 100 μg/ml proteinaseK and 0.5% SDS as stated in the manual from the Supercos 1 cosmid vectorkit; the rest of the DNA preparation was done according to theinstructions of the kit.

The size of the genomic DNA was analysed by electrophoresis using theCHEF-gel apparatus (Bio-Rad Laboratories, Hercules Calif., USA). A 1%agarose gel was run for 20 hours at 200 volts with a 10-50 second pulse.The gel was stained with ethidium bromide and photographed. The DNA was50→100 kb in size. The DNA was partially digested using Sau3A.

The size of the digested DNA was 20-50 kb determined by the same type ofCHEF-gel analysis as above. The CsCl gradient banded SuperCos1 vectorwas prepared according to the manual. Ligation and packaging werelikewise performed as described in the manual.

After titration of the library, all of the packaging mix from oneligation and packaging was transfected into the host cells, XL1-Blue MR,and plated on 50 μg/ml ampicillin LB plates. Approximately 3800 colonieswere obtained. Cosmid preparations from 10 colonies showed that they allhad inserts of the expected size. The colonies were picked individuallyand inoculated in microtiter plate wells with 100 μl LB (100 μg/mlampicillin) and incubated at 37° C. overnight. 100 μl of 50% glycerolwas added to each well, and the entire library was frozen at −80° C. Atotal of 3822 colonies were stored.

This represents the A. oryzae genome approximately 4.4 times. Afterpicking the colonies the plates were scraped off, the scrape-off pooledand the total library was also stored in four pools as frozen glycerolstock. The four pools were named ToC901 -ToC904.

The individually frozen colonies in the library were inoculated ontoLB-plates (100 μg/ml ampicillin) by using a multipin device of 6 rows of8 pins fitting into half a microtiter dish. Plates were made containingcolonies from all clones in the library.

The plates were incubated at 37° C. overnight. Sterilized Whatman 540filters cut to the size of a petri dish were placed upon the colonieswhich were incubated for two more hours at 37° C. The filters weretransferred to LB plates containing 200 μg/ml of chloramphenicol and theplates were incubated overnight at 37° C.

The next day the filters were washed twice in 0.5 M NaOH for 5 minutes,then twice in 0.5 M Tris-HCl (pH7.4) for 5 minutes and then twice in2×SSC for 5 minutes. The filters were wetted with ethanol and air dried.

Selection of amyR ClonesSelection of amyR Clones

Cosmid DNA was prepared from ToC901-904 and introduced into ToC879 byco-transformation with pMT1657. The transformation procedure isdescribed in EP Application No. 87103806.3. Approximately 8700transformants were selected by resistance to 1 mg/ml glufosinate inminimal plates (Cove D. J. (1966) BBA 113 51-56) containing 1 M sucrosefor osmotic stabilization and 10 mM (NH₄)₂SO₄.

Ten randomly chosen transformants were reisolated once on the same typeof plates. Conidiospores from these 10 transformants were inoculated inminimal medium containing 1 mg/ml glufosinate and grown at 30° C. untilenough mycelium for DNA preparation could be harvested. DNA was preparedas described in T. Christensen et al. (supra).

The uncut DNA was applied to a 0.7% agarose gel, and electrophoresis wasperformed, followed by Southern blotting. The blot was hybridized with a³²P-labelled SuperCos1 specific DNA fragment. Each of the tentransformants showed a band with a higher mobility than the linearchromosomal DNA. Each of the bands also hybridized to a pHelp1 specificprobe, indicating that the co-transformation frequency of the cosmidlibrary was close to 100% and that the cosmids had integrated into theautonomously replication vector pHelp1.

The transformants were unstable as expected for pHelp1 transformants.Less than 10% of the conidiospores from a glufosinate resistant colonygave rise to glufosinate-resistant progeny.

Conidiospores from all the transformants were collected in 8 pools andplated on minimal plates (Cove D. J., supra) containing 1 mg/mlglufosinate, 10 mM (NH₄)₂SO₄ and 1% b-cyclodextrin (Kleptose fromRoquette Frèreś, 62136 Lestem, France)

Four colonies were obtained from one of the pools and one from one ofthe other pools. Two of the colonies from the first pool were reisolatedonce on the same kind of plates.

Conidiospores from the reisolated colonies were plated on minimal plateswith either glucose or cyclodextrin as a carbon source and onglufosinate-containing plates. The glufosinate resistance and theability to grow on cyclodextrin were both unstable phenotypes with thesame degree of instability. This indicated that the gene conferring theability to grow on cyclodextrin was physically linked to pMT1657 in thetransformants.

Colonies from the reisolation plates were cut out and were analysed byrocket immune electrophoresis (RIE) using an antibody raised against theHLL lipase. The transformants gave a clear reaction with the antibody,while ToC879 colonies grown on maltose gave no reaction. This led to theconclusion that both the expression of amylase (i.e., growth oncyclodextrin) and lipase (i.e. antibody binding) had been restored inthese transformants. The gene responsible for this phenotype was namedamyR.

Isolation of the amyR Gene

In order to rescue the amyR gene from the amylase⁺, lipase⁺transformants of ToC879, two different approaches were usedsuccessfully.

DNA was prepared from mycelium grown in minimal medium with cyclodextrinas the carbon source.

In the first approach the DNA was packaged into λ-heads using theGigapack® II kit from Stratagene in an attempt to rescue the originalcosmid out of the total DNA. The packaging reaction was incubated withXL1-Blue MR E. coli under the conditions specified by the kit supplier.The E. coli cells were plated on LB plates with 50 μg/ml ampicillin. Twocolonies appeared on the plates; the cosmids they contained wereidentical and named ToC1012.

In the second approach the total DNA was used in an attempt to transformcompetent E. coli DH5a cells. Sixteen colonies were isolated and shownto contain six different plasmids by restriction enzyme digest. Each ofthe plasmids was digested with EcoRI and subjected to Southern analysis.A ³²P-labelled probe of a mixture of pMT1657 and SuperCos1 was used toidentify DNA fragments not part of any of these vectors. Two EcoRIfragments, approximately 0.7 and 1.2 kb in size, did not hybridize toany of these probes. The 1.2 kb fragment was isolated, labelled with ³²Pand used as a probe in a hybridization experiment with the filterscontaining the part of the cosmid library that gave rise to the originaltransformants. Six cosmids from the pool (ToC904), containingapproximately 1000 clones did hybridize.

Of these, some were shown by restriction enzyme digestion to beidentical, resulting in the isolation of four different cosmids. Allcosmids contained at least parts of the TAKA-amylase gene as well. Thefour cosmids and the cosmid ToC1012 were transformed into ToC879 byco-transformation with pMT1623, a pUC based plasmid that carries the bargene under the control of the A. oryzae tpi promoter. Fifteentransformants from each co-transformation were isolated by resistance toglufosinate and tested for the ability to grow on cyclodextrin.

Eight transformants of ToC1012 and three transformants of one of theother cosmids, 41B12, were able to grow. None of the transformants ofthe other cosmids grew. That not all of the transformants of ToC1012 and41B12 were able to grow is likely to be a reflection of theco-transformation frequency in each experiment. Colonies from thetransformants growing on cyclodextrin were analysed by RIE, and showedthat they all produced lipase.

DNA fragments obtained by digesting 41 B12 with either BgIll, HindIII orPstI were cloned into pUC19 in order to subclone amyR from the cosmid.The subclones were transformed into ToC879 and the transformantsanalysed for the ability to grow on cyclodextrin and produce lipase asdescribed above. As depicted in FIG. 2, one plasmid called pToC316 wasshown to contain an approximate 9 kb HindIII fragment which wasidentified as containing amyR.

Further subcloning resulted in a plasmid called pToC320 containing a 4.3kb HindIII/SacI fragment, which is shown in FIG. 3 and was subsequentlysequenced on an ABI DNA sequencer using both further subcloning andprimer walking.

A DNA sequence of 3980 bp including the amyR gene is shown in SEQ IDNO: 1. The deduced amino acid sequence is shown in SEQ ID NO: 3 andreveals a Gal 4-type zinc finger sequence between amino acids 28-54.Such sequences are known to bind to DNA (Reece, R. J., and Ptashne, M.(1993) Science 261 909-910).

amyR maps close to one of the three amylase genes in IFO4177, since itwas isolated from a cosmid also containing amylase-specific DNAfragments. Mapping of the cosmid showed that the α-amylase gene and amyRare 5-6 kb apart. Southern analysis of genomic DNA showed that only onecopy of amyR is present in IFO4177, and confirmed that it maps close toone of the amylase genes.

Analysis of amyR cDNA

mRNA was made by the method of Wahleithner, J. A., et al. (1996, Curr.Genet 29 395-403) from a culture of A. oryzae grown in maltosecontaining medium under conditions favorable for α-amylase production.Double stranded cDNA was made by standard procedures and used for PCRreactions with the following primers:

oligodT primer: TTTTGTAAGCT₃₁ SEQ ID NO: 9 23087:CCCCAAGCTTCGCCGTCTGCGCTGCTGCCG SEQ ID NO: 6 20865:CGGAATTCATCAACCTCATCAACGTCTTC SEQ ID NO: 7 20866:CGGAATTCATCGGCGAGATAGTATCCTAT SEQ ID NO: 8

A PCR reaction with the primers 20866 and 23087 resulted in a fragmentof approximately 1.1 kb. The fragment was digested with EcoRI andHindIII; these restriction sites were incorporated into the primers, andcloned into a pUC19 vector cut with the same enzymes.

The insert in the resulting plasmid was sequenced, the result locatedone intron in this part of the gene. The intron is indicated in SEQ IDNO: 2.

Another PCR reaction with the oligodT primer and primer 20866 did notresult in a distinct fragment. An aliquot of this reaction was used asthe starting point for a new reaction with the oligodT primer and theprimer 20865, which resulted in a fragment of approximately 1.1 kb. Thisfragment was digested with EcoRI and HindIII and cloned into pUC19.

Sequencing showed that the fragment contained the 3′ part of amyR andanother intron was located. This is also indicated in SEQ ID NO: 2.Three independent plasmids were sequenced at the 3′ end and two polyAaddition sites were located, one at bp no. 3827 and one at bp no. 3927.

Example 2

Quantification of Glucoamylase Synthesis in an amyR Strain

A. oryzae produces a glucoamylase, encoded by the gIaA gene, which isregulated by the same substances as α-amylase (Y. Hata et al.(1992)Curr. Genet. 22 85-91). In order to see whether amyR is also involved inregulation of gIaA the synthesis of glucoamylase was measured underinducing conditions in the amyR strain ToC879 and in the amyR wt strainSRe440, from which ToC879 was directly derived.

Conidiospores from each strain were inoculated in 10 ml YPM (YPcontaining 2% maltose) and grown for four days at 30° C. Supernatantswere collected and analysed for glucoamylase content by incubation withp-nitrophenyl a-D-glucopyranoside, a substrate that turns yellow whencleaved by glucoamylase. In the procedure used, 0.5 ml of fermentationbroth was mixed with 1 ml of 0.1 M Na-acetate pH=4.3, containing 1 mg/mlof the substrate. The samples were incubated for 3 hours at roomtemperature and 1.5 ml of 0.1 M Na₂B₄O₇ was added. The yellow colour wasmeasured in a spectrophotometer at 400 nm. Control samples were made bymixing the supernatants first with the borate and then with thesubstrate solution. The results were:

reaction-control (OD units) SRe440 0.655 ToC879 0.000

The absence of any OD reading in the sample taken from ToC879 clearlyindicate that synthesis of glucoamylase of A. oryzae requires theexpression of the AmyR transcription factor.

Example 3

Overexpression of AmyR

A plasmid, pToC342, containing the coding region and 3′ noncodingsequences of amyR fused to the promoter for the A. oryzae tpi gene wasconstructed. The tpi gene codes for triosephosphate isomerase, aconstitutively expressed enzyme involved in primary metabolism. The A.oryzae tpi gene was isolated by crosshybridization with an A. nidulanscDNA clone according to the procedure of McKnight, G. L., et al, (1986,Cell 46 143-147). Sequencing led to identification of the structuralgene. The promoter used was a fragment of approximately 700 bpimmediately upstream of the coding region. pToC342 was able tocomplement the mutation in ToC879. To pToC342 was further added the A.oryzae pyrG gene and the resulting plasmid, pToC359, was transformedinto JaL250, a pyrG mutant of JaL228 described in patent applicationDK1024/96 filed Sep. 19, 1996. Strains containing multiple copies ofpToC359 were found to synthesise increased levels of glucoamylase.

Construction of pToC342 and pToC359

A PCR reaction was made with pToC320 as the template and the followingprimers:

8753 GTTTCGAGTATGTGGATTCC (SEQ ID NO:10) 8997CGGAATTCGGATCCGAGCATGTCTCATTCTC (SEQ ID NO:11)

The resulting fragment was cut with EcoRilApal to produce a fragment ofapproximately 180 bp which was then cloned into pToC320 that had beendigested with EcoRIIApaI. The resulting plasmid, pToC336, was sequencedto confirm that the PCR fragment was intact. The 2.6 kb BamHI/SacIfragment of pToC336 containing the coding region and the 3′ untranslatedsequence of amyR and an EcoRI/BamHI fragment of approximately 700 bpcontaining the tpi promoter was cloned into EcoRI/SacI digested pUC19.The BamHI site downstream of the tpi promoter was introduced in vitro,whereas the EcoRI site is an endogenous site from the original tpiclone. The resulting plasmid, called pToC342, was cut with HindIII,dephosphorylated and ligated to a 1.8 kb HindIII fragment containing theA. oryzae pyrG gene, resulting in a plasmid which was called pToC359.The structure of both pToC342 and pToC359 are shown in FIG. 4, whereinPtpi represents the tpi promoter and TamyR represents the 3′ noncodingregion of amyR. The cloning of the pyrG gene has been previouslydescribed in WO 95/35385.

Expression in A. oryzae JaL250

JaL250 is a pyrG mutant of JaL228 selected by resistance to5-fluoro-orotic acid. JaL228 has been described in patent applicationDK1024/96 filed Sep. 19, 1996. JaL250 was transformed with pToC359 usingstandard procedures and by selecting for relief of uridine requirement.The transformants were reisolated twice through conidiospores and grownfor four days in YP+2% maltose at 30° C. Secreted glucoamylase wasmeasured by the ability to cleave p-nitrophenyl a-D-gluco-pyranoside.The transformants had 5-31 arbitrary glucoamylase units/ml in thefermentation broth, while JaL228 had 2-3 units/ml. The best transformantwas named ToC1200. Southern analysis showed that multiple copies ofpToC359 had integrated into the genome of ToC1200. Because of thealpha-amylase promoter, ToC1200 may be used advantageously as a hoststrain for expression plasmids.

Example 4

Carbon Catabolite Repression of the TAKA-promoter

The TAKA-amylase promoter is subject to carbon catabolite repression. InAspergilli carbon catabolite repression is at least partially mediatedvia the transcriptional repressor CreA, a homologue to S. cerevisiaeMIG1. The DNA binding sites in promoters under CreA control are known tobe GC-rich and seemingly identical to the MIG1 sites in S. cerevisiae.The TAKA-amylase promoter contains several potential CreA binding sites.To determine whether this promoter is involved in carbon cataboliterepression, three such sites were mutated, but provided only partialrelief of carbon catabolite repression. In contrast, introduction ofcopies of constitutively expressed AmyR in strains containing themodified promoter coupled to a reporter gene completely relievedrepression of the reporter.

Construction of a CreA Site Deleted TAKA-amylase Promoter

Three sites were identified as being potential CreA binding sites in theTAKA-amylase promoter by sequence comparison to known CreA and MIG1sites. The resulting sites have the following sequences:

Site I CCCCGGTATTG (SEQ ID NO: 12) Site II CCCCGGAGTCA (SEQ ID NO: 13)Site III ATATGGCGGGT (SEQ ID NO: 14)

The bases underlined were changed to A's because such changes are knownto destroy MIG1 binding sites. The substitutions were made usingstandard site-specific mutagenesis procedures. An expression vector,pToC297, containing the modified promoter and the 3′ nontranscribedsequence of the glucoamylase gene from A. niger was constructed. pToC297is identical to pToC68 described in WO 91/17243 except for the changesin the promoter. Both plasmids have a unique BamHI site between thepromoter and the terminator.

Expression of a Lipase Regulated by a CreA TAKA-amylase Promoter

A BamHI fragment of approximately 950bp containing the cDNA encoding aHumicola lanuginosa lipase was cloned into pToC297. (The cloning andexpression of the H. lanuginosa lipase has been previously described inEP 305 216.) The resulting plasmid, pToC298, was transformed into A.oryzae IF04177 by co-transformation with the A. nidulans amdS gene, andits structure is shown in FIG. 5, wherein Ptaka-creA represents the CreAbinding site deficient TAKA-amalyase promoter. The transformants werereisolated twice through conidiospores and one such transformant,ToC1075, which produces lipase, was chosen for further evaluation.ToC1075 and a p960 transformant of IFO4177 (previously described in EP305 216) containing the lipase fused to the wild type TAKA-promoter weregrown at 30° C. in 10 ml YP containing 2% or 10% glucose. Samples weretaken daily for analysis of lipase in the fermentation broth. The lipasecontent was measured by rocket immune electrophoresis using a polyclonalantibody raised against purified lipase. Spent fermentation broth fromA. oryzae IF04177 did not react with the antibody. The glucose contentof the fermentation broth was likewise measured daily using Tes-tapefrom Lilly.

On day one, glucose was detected in all cultures, but on day two glucosecould be detected only in cultures originally containing 10%. Theresults of lipase production, shown in FIG. 6, indicate that the wildtype promoter is repressed until glucose is no longer present. Thus,when the glucose becomes exhausted, lipase begins to accumulate. FIG. 6also shows that the modified promoter is not as tightly regulated, aslow levels of lipase are produced in the presence of glucose in the 10%glucose fermentation. Thus, there is partial glucose derepression seenin ToC1075.

Relief of Carbon Catabolite Repression of Lipase in ToC1075 by pToC342

ToC1075 was transformed with pToC342 by co-transformation with thebar-containing plasmid, pMT1623. Strains containing multible copies ofpToC342 and which retained the lipase expression cassette wereidentified by Southern blot analysis; one such strain was. ToC1075 andToC1139 were grown at 30° C. in 10 ml YP containing either 2% or 10%glucose, and samples were assayed daily for lipase and glucose. Thelipase was measured by cleavage of paranitrophenyl-butyrate. The glucosecontent was measured with Tes-tape from Lilly. The results, shown inFIG. 7, indicate that ToC1075, as before, provides partial relief ofglucose repression while lipase production by ToC1139 is independent ofthe presence of glucose.

Example 5

Southern Analysis of A. niger for the amyR Gene

The syntheses of alpha-amylase and glucoamylase in A. niger, as in A.oryzae, are regulated by the carbon source. It is therefore likely thatA. niger also contains an amyR gene. This hypothesis was tested bylooking for cross-hybridization between the A. oryzae amyR gene and A.niger chromosomal DNA.

DNA was prepared from A. niger by conventional methods. The DNA was cutwith BamHI, BgiII, EcoRI, HindIII, SaII, XmaI or XbaI, and the resultingDNA fragments were separated by electrophoresis on an agarose gel. TheDNA was then blotted onto a nitrocellulase membrane and hybridized witha ³²P-labelled probe containing part of the structural A. oryzae amyRgene. The probe was made by PCR on pToC320 and starts at bp. no. 1683and ends at bp. no. 2615 as shown in SEQ ID NO: 1. The hybridization wasconducted in 10×Denhardt's solution, 5×SSC, 10 mM EDTA, 1% SDS, 0.15mg/ml polyA, 0.05 mg/ml yeast tRNA) at 50° C. overnight. Afterhybridization the membrane was washed under conditions of increasingstringency and the radioactivity on the membrane analysed by aPhosphoImager. FIG. 8 shows the result when the membrane had been washedin 2×SSC, 0.1%SDS at 58° C. Unique bands can be seen with several of therestriction enzymes. Thus, the A. niger amyR gene can be cloned on thebasis of this cross-hybridization result.

REFERENCES CITED IN THE SPECIFICATION

Description

Dhawale and Lane (1993) NAR 21 5537-5546

Lachmund et al. (1993) Current Microbiology 26 47-51

Tada et al. (1991) Mol. Gen. Genet 229 301-306

Tada et al. (1991) Agric. Biol. Chem. 55 1939-1941

Tsuchiya et al. (1992) Biosci. Biotech. Biochem. 56 1849-1853,

Nagata et al. (1993) Mol. Gen. Genet 237 251-260

Ford et al., (1991), Protein Expression and Purification 2, 95-107

Cunningham and Wells, (1989) Science 244 1081-1085

Needleman, S. B. and Wunsch, C. D., (1970) Journal of MolecularBiology48 443-453

Sambrook, J., et al., (1989), Molecular Cloning: A Laboratory Manual.Cold Spring Harbor Lab.; Cold Spring Harbor, N.Y.

WO 93/11249

WO 94/14953

Kelly and Kwon-Chung (1992) J. Bacteriol. 174 222-232

McKnight et al. (1985) The EMBO J. 4 2093-2099

EP 238 023

Kulmburg, P., et al. (1992) Molecular and Cellular Biology 12 1932-1939

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Examples

Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem. 132:6-13

N. Axelsen et al. in: A Manual of Quantitative Immunoelectrophoresis,Blackwell Scientific Publications, 1973, Chapter 23

A. Johnstone and R. Thorpe, Immunochemistry in Practice, BlackwellScientific Publications, 1982, pp. 27-31

O. Ouchterlony in: Handbook of Experimental Immunology (D. M. Weir,ed.), Blackwell Scientific Publications, 1967, pp. 655-706

D. Gems, I. L. Johnstone, and A. J. Clutterbuck, (1991) Gene 98 61-67

EP 531 372

B. Staubinger et al. (1992) Fungal Genetics Newsletter 39 82-83

M. E. Katz et al. (1990) Mol. Gen. Genet. 220 373-376

Christensen, T., et al., (1988) Biotechnology 6 1419-1422

Cove D. J., BBA (1966) 113 51-56

Reece, R. J., and Ptashne, M. (1993) Science 261 909-910

Wahleithner, J. A., et al. (1996) Curr. Genet. 29 395-403

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McKnight, G. L., et al, (1986, Cell 46 143-147)

DK1024/96

WO 95/35385

WO 91/17243

EP 305 216

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 14 <210> SEQ ID NO 1 <211> LENGTH: 3980<212> TYPE: DNA <213> ORGANISM: Aspergillus oryzae <400> SEQUENCE: 1tctagaccgg ccatgtcgtg gtccgccaag ttgattccgg accgtgttgt ag#ttgcttct     60tttaagaaac ggcacccctc tgccgtctcc gaaccggaat tgtagctaga tg#tatatgtc    120ttgacgaacc aggtgtccac gggcaaatcc ctcacaattg atggcccgtc cc#gttcccat    180cgatttgtgc tacctgccgt gcaaggcaaa acatccccgt caaacgtccg ag#gggcattg    240cctgcaatct ctcgaccatg agaggggaag caagtcacgc tagttgcaag gg#tataggtc    300ctacgcagca atgaggtggc ttcacccgta cggagtgggg acagcatgat ca#agcctttt    360gggaacgtga cgaaagagta ccggttaagc cgacgatggg agatgaatct ct#gccgagca    420aaggacgaga ccggaaaaga gtgtgttgat tcttgggagc agttacagta ct#tccgtgtc    480cggaaattgg aaacgttcct gaccaatgct ggcgatcatc tgatatccct ac#gctgattg    540gtccatcccc cgataaatgc ccgacacgac gcttgagccc tgaaaaggta gt#atttctcg    600agagatccat tcaccagagt caatactggc aaatacatcg ttccccacct ca#tattccaa    660ggtgcctaaa cccctccggt gtgccggtga gggttttcca cgccatctct ag#tggtgcca    720tgacgggagc atccgatggc ttccagtatt gggtggttgg gatggacaac aa#gctccaaa    780taaggggaat ttgcctttgg tccaggaatg aagtccccgt ggggaccagc gg#ctcagccc    840aggctaagag tggaatatcg tcatagacct tcggctcatg ggaggttcgg ag#gtgttacg    900atcctcttca atgccattca ttctctgttt tgacctcggc ttcccgagag tg#gtgcctcc    960cttacatccc cacatgctgg atgcaagcct gtggtacgct gtttctttca ga#agtagcag   1020gctaggttca cgatgagctg cctttcaaac ctggaataac cattacgtga ga#ctgttcta   1080cttcttgaat tgatccctga ctagagtctg ctctaatatg ctgtgtggca cg#gccggtcc   1140cctcggggtt gctaaggctg atttatgcac tccgtacagt ataacccagg gt#ggctatag   1200attccctgca tcttccacgc tccctcacaa cctgattcca ccattcttaa gc#ggccgtta   1260gcctcgatgg ggtataatgg agttaactat aaacacgact ctacaacgaa tc#ccgatgtg   1320agtttggaac gagttgttac cgatgggtcc tcccatttgt taggagtgac gc#taggggac   1380ctttagggca cagactaaac caagacaaag atggagtaga ctccaggtag at#taattcca   1440atcttcttgc caaagtaacg cggggttttt tgcacctgca gcctcttttt tt#tctttttt   1500cttttttttc tttttttatt gttccccaga tttcttttct ttttcttcaa tc#ctgacgtt   1560ctcaaccgtg atggcgacac agcccgcttc gctatccctc gcttttacgt cg#gccattct   1620tctagttgct ctcgcgggat gccatgattt ctaaaggctc cacatcggcg ag#atagtatc   1680ctatccgagc atgtctcatt ctccaaccga cattccctca acatccgaaa ag#gaaatgga   1740gtcaacccca gaaaagccgc ctaaacaggc ctgcgacaat tgccgtcgac gc#aaaatcaa   1800gtgttctaga gagcttccat gcgacaagtg ccagcgtctt cttctctcct gt#tcctacag   1860cgacgtgctc cgtcgcaagg gccccaagtt ccgcacgctc taccctctcg ct#cccatcca   1920tccactcgcc tcacgaccac gtcctctcac caaggaatgg ctgcccccaa ac#ccaggggc   1980ttgccatttg gcgtccccga cgtctccgcc gtccaccgta gcggacgccc ag#tatctaca   2040tccagacttc tcggagtcgt tcactcgact accaccccca gatctcgtct cc#tctcccga   2100ctcgacaaac tcgctattcg actcgtccac tatcggcgca ctccccgcgc ca#cgccgtct   2160gtcgacgcca aaccttctag cccatgtcaa tgtcttcctc aagtacctgt tc#ccgatcat   2220gcccgtcgtg agacaggacc agctgcagca ggactgccac cagccggagc gc#ttgtctcc   2280ccaacgctac gctttcattg ccgctctatg cgcggccacg cacatccaac tg#aagctgga   2340cggtgcagca ccgggtcccg aggcggcttc cgcgcgagcc agcctcgacg ga#catcctat   2400gttgtcggga gaagaactcc tggctgaagc cgtgcgcgca agaaaggaat ac#aacgtggt   2460cgacgaaatt aacatggaaa acctcctaac ctccttcttt ctcttcgccg cc#tacggaaa   2520cctagacaga caggatcagg cctggttcta cctatgtcag accacgtcca tg#gtcttcac   2580actaggccta caacgggaat ccacatactc gaaactaagc gtcgaggaag ca#gaagagaa   2640aaggagagta ttctggctct tattcgtcac agaaaggtaa gaaaagaaaa aa#ctctactt   2700tcccaatcac caccacgtac caaaaataac acgaaaaacc agaggctacg ca#ttacaaca   2760agcaaaacca gtcatgctcc gcaactccat ccacaaacca caggtcctgt gc#tcagacga   2820cccaatccta gcctacgggt tcatcaacct catcaacgtc ttcgaaaagc tc#agcccaaa   2880tctctacgac tgggtctccg ccggcggcag cagcgcagac ggcgaccccc cg#cctacttc   2940ttctatccaa tccagtctcg ccaagcaaat ctccctcgag ggcgtctccg ag#atccagaa   3000agtagacatc ctcatcactc agcaatggct acaaaccatg atgtggaaac tc#tccatgac   3060ccacgtcaca cagcccggct ctcgcgatga cgccgttctc cccttccacc tg#cccgtgct   3120agtcggcaag gccgtcatgg gcgtcatcgc cgcggcatcc caaggtgctg tt#gacgctca   3180tggtatcgga atggtaagaa agcgacctta cctcatcaca ccctccctca tc#agtcactc   3240cccatcatct atacccgcaa tctaacaaaa accgcaggaa caaaaactct ac#gacctcgg   3300cacctccgta gccgacgtct cccgctccct aagcacaaaa gccgcccacc ac#ctcgccga   3360atcgaccatc gacccccgag aactcctctg gggcattctc acaaccctat cc#cgaatccg   3420cggttcccaa tcatacctct tcccagcgct cgtcgagcaa agtcgaggca tc#atcagttt   3480cgactgttcg ctttccatca gtgactttct gccttcgttt ggtgggccgc cg#gctattat   3540gtggcggacg ggtgaatctg ggtttgattt attggggatc gcggatgatt tg#caagagag   3600ggagaatgag ggtggggagg ggattgtggt ggctggggag gagatttcgt tt#tgaggggg   3660ctcttttctt tttcctttgt ggtgtgttgt gttgggtggt tctggggggg cg#ggggtgta   3720tatacgcttg acgatgtgca ttgggattgg ggttcctact ggtatataat at#ggattgtt   3780ttgtatatag tccgctggag acggtgcaat gatgtgggga tcaatcactt ct#taggactc   3840ggagcacagg gtgtcggttc tcgggttatt ctgagtatga gattatatag aa#tcagttaa   3900tgatcattat tgtacatacc ttaaagaaag atatgcttgg caccccgata tg#acaataga   3960 aaactggtct tcattctaga             #                  #                 398 #0 <210> SEQ ID NO 2 <211> LENGTH: 3980<212> TYPE: DNA <213> ORGANISM: Aspergillus oryzae <400> SEQUENCE: 2tctagaccgg ccatgtcgtg gtccgccaag ttgattccgg accgtgttgt ag#ttgcttct     60tttaagaaac ggcacccctc tgccgtctcc gaaccggaat tgtagctaga tg#tatatgtc    120ttgacgaacc aggtgtccac gggcaaatcc ctcacaattg atggcccgtc cc#gttcccat    180cgatttgtgc tacctgccgt gcaaggcaaa acatccccgt caaacgtccg ag#gggcattg    240cctgcaatct ctcgaccatg agaggggaag caagtcacgc tagttgcaag gg#tataggtc    300ctacgcagca atgaggtggc ttcacccgta cggagtgggg acagcatgat ca#agcctttt    360gggaacgtga cgaaagagta ccggttaagc cgacgatggg agatgaatct ct#gccgagca    420aaggacgaga ccggaaaaga gtgtgttgat tcttgggagc agttacagta ct#tccgtgtc    480cggaaattgg aaacgttcct gaccaatgct ggcgatcatc tgatatccct ac#gctgattg    540gtccatcccc cgataaatgc ccgacacgac gcttgagccc tgaaaaggta gt#atttctcg    600agagatccat tcaccagagt caatactggc aaatacatcg ttccccacct ca#tattccaa    660ggtgcctaaa cccctccggt gtgccggtga gggttttcca cgccatctct ag#tggtgcca    720tgacgggagc atccgatggc ttccagtatt gggtggttgg gatggacaac aa#gctccaaa    780taaggggaat ttgcctttgg tccaggaatg aagtccccgt ggggaccagc gg#ctcagccc    840aggctaagag tggaatatcg tcatagacct tcggctcatg ggaggttcgg ag#gtgttacg    900atcctcttca atgccattca ttctctgttt tgacctcggc ttcccgagag tg#gtgcctcc    960cttacatccc cacatgctgg atgcaagcct gtggtacgct gtttctttca ga#agtagcag   1020gctaggttca cgatgagctg cctttcaaac ctggaataac cattacgtga ga#ctgttcta   1080cttcttgaat tgatccctga ctagagtctg ctctaatatg ctgtgtggca cg#gccggtcc   1140cctcggggtt gctaaggctg atttatgcac tccgtacagt ataacccagg gt#ggctatag   1200attccctgca tcttccacgc tccctcacaa cctgattcca ccattcttaa gc#ggccgtta   1260gcctcgatgg ggtataatgg agttaactat aaacacgact ctacaacgaa tc#ccgatgtg   1320agtttggaac gagttgttac cgatgggtcc tcccatttgt taggagtgac gc#taggggac   1380ctttagggca cagactaaac caagacaaag atggagtaga ctccaggtag at#taattcca   1440atcttcttgc caaagtaacg cggggttttt tgcacctgca gcctcttttt tt#tctttttt   1500cttttttttc tttttttatt gttccccaga tttcttttct ttttcttcaa tc#ctgacgtt   1560ctcaaccgtg atggcgacac agcccgcttc gctatccctc gcttttacgt cg#gccattct   1620tctagttgct ctcgcgggat gccatgattt ctaaaggctc cacatcggcg ag#atagtatc   1680ctatccgagc atgtctcatt ctccaaccga cattccctca acatccgaaa ag#gaaatgga   1740gtcaacccca gaaaagccgc ctaaacaggc ctgcgacaat tgccgtcgac gc#aaaatcaa   1800gtgttctaga gagcttccat gcgacaagtg ccagcgtctt cttctctcct gt#tcctacag   1860cgacgtgctc cgtcgcaagg gccccaagtt ccgcacgctc taccctctcg ct#cccatcca   1920tccactcgcc tcacgaccac gtcctctcac caaggaatgg ctgcccccaa ac#ccaggggc   1980ttgccatttg gcgtccccga cgtctccgcc gtccaccgta gcggacgccc ag#tatctaca   2040tccagacttc tcggagtcgt tcactcgact accaccccca gatctcgtct cc#tctcccga   2100ctcgacaaac tcgctattcg actcgtccac tatcggcgca ctccccgcgc ca#cgccgtct   2160gtcgacgcca aaccttctag cccatgtcaa tgtcttcctc aagtacctgt tc#ccgatcat   2220gcccgtcgtg agacaggacc agctgcagca ggactgccac cagccggagc gc#ttgtctcc   2280ccaacgctac gctttcattg ccgctctatg cgcggccacg cacatccaac tg#aagctgga   2340cggtgcagca ccgggtcccg aggcggcttc cgcgcgagcc agcctcgacg ga#catcctat   2400gttgtcggga gaagaactcc tggctgaagc cgtgcgcgca agaaaggaat ac#aacgtggt   2460cgacgaaatt aacatggaaa acctcctaac ctccttcttt ctcttcgccg cc#tacggaaa   2520cctagacaga caggatcagg cctggttcta cctatgtcag accacgtcca tg#gtcttcac   2580actaggccta caacgggaat ccacatactc gaaactaagc gtcgaggaag ca#gaagagaa   2640aaggagagta ttctggctct tattcgtcac agaaaggtaa gaaaagaaaa aa#ctctactt   2700tcccaatcac caccacgtac caaaaataac acgaaaaacc agaggctacg ca#ttacaaca   2760agcaaaacca gtcatgctcc gcaactccat ccacaaacca caggtcctgt gc#tcagacga   2820cccaatccta gcctacgggt tcatcaacct catcaacgtc ttcgaaaagc tc#agcccaaa   2880tctctacgac tgggtctccg ccggcggcag cagcgcagac ggcgaccccc cg#cctacttc   2940ttctatccaa tccagtctcg ccaagcaaat ctccctcgag ggcgtctccg ag#atccagaa   3000agtagacatc ctcatcactc agcaatggct acaaaccatg atgtggaaac tc#tccatgac   3060ccacgtcaca cagcccggct ctcgcgatga cgccgttctc cccttccacc tg#cccgtgct   3120agtcggcaag gccgtcatgg gcgtcatcgc cgcggcatcc caaggtgctg tt#gacgctca   3180tggtatcgga atggtaagaa agcgacctta cctcatcaca ccctccctca tc#agtcactc   3240cccatcatct atacccgcaa tctaacaaaa accgcaggaa caaaaactct ac#gacctcgg   3300cacctccgta gccgacgtct cccgctccct aagcacaaaa gccgcccacc ac#ctcgccga   3360atcgaccatc gacccccgag aactcctctg gggcattctc acaaccctat cc#cgaatccg   3420cggttcccaa tcatacctct tcccagcgct cgtcgagcaa agtcgaggca tc#atcagttt   3480cgactgttcg ctttccatca gtgactttct gccttcgttt ggtgggccgc cg#gctattat   3540gtggcggacg ggtgaatctg ggtttgattt attggggatc gcggatgatt tg#caagagag   3600ggagaatgag ggtggggagg ggattgtggt ggctggggag gagatttcgt tt#tgaggggg   3660ctcttttctt tttcctttgt ggtgtgttgt gttgggtggt tctggggggg cg#ggggtgta   3720tatacgcttg acgatgtgca ttgggattgg ggttcctact ggtatataat at#ggattgtt   3780ttgtatatag tccgctggag acggtgcaat gatgtgggga tcaatcactt ct#taggactc   3840ggagcacagg gtgtcggttc tcgggttatt ctgagtatga gattatatag aa#tcagttaa   3900tgatcattat tgtacatacc ttaaagaaag atatgcttgg caccccgata tg#acaataga   3960 aaactggtct tcattctaga             #                  #                 398 #0 <210> SEQ ID NO 3 <211> LENGTH: 604<212> TYPE: PRT <213> ORGANISM: Aspergillus oryzae <400> SEQUENCE: 3Met Ser His Ser Pro Thr Asp Ile Pro Ser Th #r Ser Glu Lys Glu Met 1               5   #                10   #                15Glu Ser Thr Pro Glu Lys Pro Pro Lys Gln Al #a Cys Asp Asn Cys Arg            20       #            25       #            30Arg Arg Lys Ile Lys Cys Ser Arg Glu Leu Pr #o Cys Asp Lys Cys Gln        35           #        40           #        45Arg Leu Leu Leu Ser Cys Ser Tyr Ser Asp Va #l Leu Arg Arg Lys Gly    50               #    55               #    60Pro Lys Phe Arg Thr Leu Tyr Pro Leu Ala Pr #o Ile His Pro Leu Ala65                   #70                   #75                   #80Ser Arg Pro Arg Pro Leu Thr Lys Glu Trp Le #u Pro Pro Asn Pro Gly                85   #                90   #                95Ala Cys His Leu Ala Ser Pro Thr Ser Pro Pr #o Ser Thr Val Ala Asp            100       #           105       #           110Ala Gln Tyr Leu His Pro Asp Phe Ser Glu Se #r Phe Thr Arg Leu Pro        115           #       120           #       125Pro Pro Asp Leu Val Ser Ser Pro Asp Ser Th #r Asn Ser Leu Phe Asp    130               #   135               #   140Ser Ser Thr Ile Gly Ala Leu Pro Ala Pro Ar #g Arg Leu Ser Thr Pro145                 1 #50                 1 #55                 1 #60Asn Leu Leu Ala His Val Asn Val Phe Leu Ly #s Tyr Leu Phe Pro Ile                165   #               170   #               175Met Pro Val Val Arg Gln Asp Gln Leu Gln Gl #n Asp Cys His Gln Pro            180       #           185       #           190Glu Arg Leu Ser Pro Gln Arg Tyr Ala Phe Il #e Ala Ala Leu Cys Ala        195           #       200           #       205Ala Thr His Ile Gln Leu Lys Leu Asp Gly Al #a Ala Pro Gly Pro Glu    210               #   215               #   220Ala Ala Ser Ala Arg Ala Ser Leu Asp Gly Hi #s Pro Met Leu Ser Gly225                 2 #30                 2 #35                 2 #40Glu Glu Leu Leu Ala Glu Ala Val Arg Ala Ar #g Lys Glu Tyr Asn Val                245   #               250   #               255Val Asp Glu Ile Asn Met Glu Asn Leu Leu Th #r Ser Phe Phe Leu Phe            260       #           265       #           270Ala Ala Tyr Gly Asn Leu Asp Arg Gln Asp Gl #n Ala Trp Phe Tyr Leu        275           #       280           #       285Cys Gln Thr Thr Ser Met Val Phe Thr Leu Gl #y Leu Gln Arg Glu Ser    290               #   295               #   300Thr Tyr Ser Lys Leu Ser Val Glu Glu Ala Gl #u Glu Lys Arg Arg Val305                 3 #10                 3 #15                 3 #20Phe Trp Leu Leu Phe Val Thr Glu Arg Gly Ty #r Ala Leu Gln Gln Ala                325   #               330   #               335Lys Pro Val Met Leu Arg Asn Ser Ile His Ly #s Pro Gln Val Leu Cys            340       #           345       #           350Ser Asp Asp Pro Ile Leu Ala Tyr Gly Phe Il #e Asn Leu Ile Asn Val        355           #       360           #       365Phe Glu Lys Leu Ser Pro Asn Leu Tyr Asp Tr #p Val Ser Ala Gly Gly    370               #   375               #   380Ser Ser Ala Asp Gly Asp Pro Pro Pro Thr Se #r Ser Ile Gln Ser Ser385                 3 #90                 3 #95                 4 #00Leu Ala Lys Gln Ile Ser Leu Glu Gly Val Se #r Glu Ile Gln Lys Val                405   #               410   #               415Asp Ile Leu Ile Thr Gln Gln Trp Leu Gln Th #r Met Met Trp Lys Leu            420       #           425       #           430Ser Met Thr His Val Thr Gln Pro Gly Ser Ar #g Asp Asp Ala Val Leu        435           #       440           #       445Pro Phe His Leu Pro Val Leu Val Gly Lys Al #a Val Met Gly Val Ile    450               #   455               #   460Ala Ala Ala Ser Gln Gly Ala Val Asp Ala Hi #s Gly Ile Gly Met Glu465                 4 #70                 4 #75                 4 #80Gln Lys Leu Tyr Asp Leu Gly Thr Ser Val Al #a Asp Val Ser Arg Ser                485   #               490   #               495Leu Ser Thr Lys Ala Ala His His Leu Ala Gl #u Ser Thr Ile Asp Pro            500       #           505       #           510Arg Glu Leu Leu Trp Gly Ile Leu Thr Thr Le #u Ser Arg Ile Arg Gly        515           #       520           #       525Ser Gln Ser Tyr Leu Phe Pro Ala Leu Val Gl #u Gln Ser Arg Gly Ile    530               #   535               #   540Ile Ser Phe Asp Cys Ser Leu Ser Ile Ser As #p Phe Leu Pro Ser Phe545                 5 #50                 5 #55                 5 #60Gly Gly Pro Pro Ala Ile Met Trp Arg Thr Gl #y Glu Ser Gly Phe Asp                565   #               570   #               575Leu Leu Gly Ile Ala Asp Asp Leu Gln Glu Ar #g Glu Asn Glu Gly Gly            580       #           585       #           590Glu Gly Ile Val Val Ala Gly Glu Glu Ile Se #r Phe         595          #       600 <210> SEQ ID NO 4 <211> LENGTH: 26 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer <400> SEQUENCE: 4cttgcatgcc gccaggaccg agcaag           #                  #              26 <210> SEQ ID NO 5 <211> LENGTH: 26 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer <400> SEQUENCE: 5cttggatcct ctgtgttagc ttatag           #                  #              26 <210> SEQ ID NO 6 <211> LENGTH: 30 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer <400> SEQUENCE: 6ccccaagctt cgccgtctgc gctgctgccg          #                  #           30 <210> SEQ ID NO 7 <211> LENGTH: 29 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer <400> SEQUENCE: 7cggaattcat caacctcatc aacgtcttc          #                  #            29 <210> SEQ ID NO 8 <211> LENGTH: 29 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer <400> SEQUENCE: 8cggaattcat cggcgagata gtatcctat          #                  #            29 <210> SEQ ID NO 9 <211> LENGTH: 41 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer <400> SEQUENCE: 9ttttgtaagc tttttttttt tttttttttt tttttttttt t     #                  #   41 <210> SEQ ID NO 10 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer <400> SEQUENCE: 10gtttcgagta tgtggattcc             #                  #                   # 20 <210> SEQ ID NO 11 <211> LENGTH: 31<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primers <400> SEQUENCE: 11cggaattcgg atccgagcat gtctcattct c         #                  #          31 <210> SEQ ID NO 12 <211> LENGTH: 11 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primers <400> SEQUENCE: 12ccccggtatt g                #                   #                  #       11 <210> SEQ ID NO 13 <211> LENGTH: 11 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer <400> SEQUENCE: 13ccccggagtc a                #                   #                  #       11 <210> SEQ ID NO 14 <211> LENGTH: 11 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer <400> SEQUENCE: 14atatggcggg t                #                   #                  #       11

I claim:
 1. An isolated transcription factor capable of regulatingtranscription directed by a filamentous fungus alpha-amylase-promoter ina sequence-specific manner, which comprises an amino acid sequence whichis at least 90% homologous with the amino acid sequence of SEQ ID NO: 3,when said homology is determined using GAP with a GAP creation penaltyof 3.0 and a GAP extension penalty or a fragment thereof.
 2. Thetranscription factor of claim 1, which comprises the amino acid sequenceof SEQ ID NO: 3 or a fragment thereof.
 3. The transcription factor ofclaim 2, which comprises the amino acid sequence of SEQ ID NO:
 3. 4. Anisolated transcription factor which exhibits activity in regulating theexpression of an alpha-amylase promoter in a filamentous fungus, whichis encoded by: (a) the transcription factor-encoding part of the DNAsequence cloned into plasmid pToC320 present in E. coli, DSM 10666, or(b) a DNA sequence which is at least 70% homologous with the DNAsequence defined in (a), when said homology is determined using GAP witha GAP creation penalty of 5.0 and a GAP extension penalty of 0.3
 5. Thetranscription factor of claim 4, wherein the DNA sequence is obtainedfrom a filamentous fungus.
 6. The transcription factor of claim 5,wherein the filamentous fungus is selected from the group consisting ofAscomycetes, Basidiomycetes Phycomycetes, Zygomycetes, and fungiimperfecti.
 7. The transcription factor of claim 5, wherein thefilamentous fungus is selected from the group consisting of Aspergillus,Fusarium, Humicola, Penicillium, and Trichoderma.
 8. The transcriptionfactor of claim 7, wherein the filamentous fungus is Fusarium.
 9. Thetranscription factor of claim 7, wherein the filamentous fungus is A.awamori, A. niger, or A. oryzae.
 10. The transcription factor of claim9, wherein the filamentous fungus is Aspergillus oryzae.
 11. Thetranscription factor of claim 10, wherein the filamentous fungus is A.oryzae, IFO4177.
 12. The transcription factor of claim 4, wherein theDNA sequence is obtained from a yeast strain.
 13. The transcriptionfactor of claim 4, wherein the DNA sequence is obtained from Eschericiacoli, DSM 10666.